Relationship between mitochondrial ROS formation catalyzed by monoamine oxidase and autophagy in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) represents the major and leading cause of morbidity and mortality in diabetic patients. This study aimed at investigating changes in the gene expression profile induced by diabetes in the cardiac tissue and whether monoamine oxidases (MAO)-dependent reactive oxygen species (ROS) generation affects the transcriptome in type 1 diabetes (T1D) hearts. Moreover, we investigated the biological and functional significance of such changes in gene expression focusing on autophagy. Gene expression profile was evaluated by microarray in the cardiac tissue from a mouse model of T1D induced by treatment with streptozotocin (STZ), while MAO involvement were evaluated through a classical pharmacological approach, using pargyline as an inhibitor for both MAO-A and MAO-B. The following groups were examined: (i) control mice, (ii) STZ-treated mice, (iii) control mice treated with pargyline, (iv) STZ mice treated with pargyline. The analysis of differentially expressed genes showed that profile of samples from STZ-mice formed a distinct group and positioned itself separately from all the other groups. Notably, the expression profile of diabetic hearts treated with pargyline was similar to the expression profile of control hearts. Importantly, pargyline treatment did not affect the gene expression profile of control mice. Subsequently, genes were clustered based on specific expression patterns and we focused on genes in which pargyline prevents changes in gene expression induced by diabetes. Chosen genes were classified according to the Gene Ontology (GO) vocabulary and only enriched definitions were considered. Most represented GO classes in downregulated genes in diabetic conditions were those for nucleus, cytoskeleton and plasma membrane. Notably, the most represented GO categories within upregulated genes were those for nucleus, mitochondrion, cytoskeleton and vacuole. Gene Set Enrichment Analysis and the leading-edge analysis showed that overexpressed genes with the highest enrichment score belong to mitochondria, extracellular matrix (ECM), and catabolic processes. Pargyline administration prevented these changes. Considering our previous studies and the transcriptomic results, we hypothesized that mitochondrial ROS formation induced by diabetes and MAO activity could affect autophagy. The increase in LC3 (microtubule-associated protein light chain 3)-II occurring in diabetic hearts was prevented by pargyline that also caused an increase in p62 levels suggesting a reduction in the autophagic flux. Further analysis of changes VIII in the autophagy flux was performed in adult mouse cardiomyocytes (AMVMs) cultured with high glucose (HG) in the absence or presence of pargyline and/or inhibitor of lysosomal degradation chloroquine. Cells cultured in presence of HG and pargyline displayed a dramatic reduction in the autophagy flux that was not affected in control. Moreover, phosphorylation of AMP-activated protein kinase showed a trend to decrease in AMVMs treated with HG and pargyline. Moreover, regarding mitochondrial dynamics, we observed increased levels of mitochondrial fission factor in cells exposed to HG. This event was dramatically reduced upon MAO inhibition. Interestingly, phosphorylation of dynamin-related protein 1 on Ser616 also showed a decreasing trend. In addition to previous studies from laboratory of Prof. Di Lisa, the present results suggest that diabetes leads to profound transcriptomic changes in STZ-treated mice heart and MAO inhibition prevents such changes affecting mitochondria, ECM, and catabolic processes. Furthermore, MAO-dependent ROS formation triggered by HG acts as a signal that leads to autophagy activation. Finally, mitochondrial fission is likely reduced upon MAO inhibition in AMVMs exposed to HG. Taken together, our results provide the first evidence that mitochondrial ROS formation and specifically MAO activity modulate cardiac autophagy in DCM.